Bispecific anti-human A-beta/human transferrin receptor antibodies and methods of use

ABSTRACT

Herein are provided bispecific anti-human A-betalhuman transferrin receptor antibodies and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 15/941,655, filed on Mar. 30, 2018, which is a continuation ofInternational Application No. PCT/EP2016/073411 filed on Sep. 30, 2016,which claims benefit of European Patent Application No. 15188064.8,filed on Oct. 2, 2015. Each of prior mentioned applications are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to bispecific antibodies against humanA-beta and human transferrin receptor, methods for their production,pharmaceutical compositions containing these antibodies, and usesthereof.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acidsequences that have been filed concurrenly herewith as sequence listingtext file “SequenceListing.txt”, file size of 68 KB, created on Jun. 16,2021. The aforementioned sequence listing is hereby incorporated byreference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

About 70% of all cases of dementia are due to Alzheimer's disease whichis associated with selective damage of brain regions and neural circuitscritical for cognition. Alzheimer's disease is characterized byneurofibrillary tangles in particular in pyramidal neurons of thehippocampus and numerous amyloid plaques containing mostly a dense coreof amyloid deposits and defused halos.

The extracellular neuritic plaques contain large amounts of apre-dominantly fibrillar peptide termed “amyloid β”, “A-beta”, “Aβ4”,“β-A4” or “Aβ”; see Selkoe, Ann. Rev. Cell Biol. 10 (1994) 373-403; KooPNAS 96 (1999) 9989-9990; U.S. Pat. No. 4,666,829; Glenner BBRC 12(1984) 1131). This amyloid is derived from “Alzheimer precursorprotein/P-amyloid precursor protein” (APP). APPs are integral membraneglycoproteins (see Sisodia PNAS 89 (1992) 6075) and areendoproteolytically cleaved within the AP sequence by a plasma membraneprotease, α-secretase (see Sisodia (1992), Joe. cit.). Furthermore,further secretase activity, in particular β-secretase and γ-secretaseactivity leads to the extracellular release of amyloid-β (A(3)comprising either 39 amino acids (Aβ 39), 40 amino acids (Aβ 40), 42amino acids (Aβ 42) or 43 amino acids (Aβ 43) (see Sinha PNAS 96 (1999)11094-1053; Price, Science 282 (1998) 1078-1083; WO 00/72880 or Hardy,TINS 20 (1997) 154).

It is of note that A-beta has several naturally occurring forms, wherebythe human forms are referred to as the above mentioned Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43. The most prominent form, Aβ42, has the amino acidsequence (starting from the N-terminus):DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 05). In Aβ41,Aβ40, Aβ39, the C-terminal amino acids A, IA and VIA are missing,respectively. In the Aβ43-form an additional threonine residue iscomprised at the C-terminus of the above depicted sequence.

The time required to nucleate Aβ40 fibrils was shown to be significantlylonger than that to nucleate Aβ42 fibrils (see e.g. Lansbury, Jr., P. T.and Harper, J. D., Ann. Rev. Biochem. 66 (1997) 385-407). As reviewed inWagner (J. Clin. Invest. 104 (1999) 1239-1332) the Aβ42 is morefrequently found associated with neuritic plaques and is considered tobe more fibrillogenic in vitro. It was also suggested that Aβ42 servesas a “seed” in the nucleation-dependent polymerization of orderednon-crystalline Aβ peptides (see e.g. Jarrett, Cell 93 (1993)1055-1058). Modified APP processing and/or the generation ofextracellular plaques containing proteinaceous depositions are not onlyknown from Alzheimer's pathology but also from subjects suffering fromother neurological and/or neurodegenerative disorders. These disorderscomprise, inter alia, Down's syndrome, hereditary cerebral hemorrhagewith amyloidosis Dutch type, Parkinson's disease, ALS (amyotrophiclateral sclerosis), Creutzfeldt Jacob disease, HIV-related dementia andmotor neuropathy.

Until now, only limited medical intervention schemes for amyloid-relateddiseases have been described. For example, cholinesterase inhibitorslike galantamine, rivastigmine or donepezil have been discussed as beingbeneficial in Alzheimer's patients with only mild to moderate disease.However, also adverse events have been reported due to cholinergicaction of these drugs. While these cholinergic-enhancing treatments doproduce some symptomatic benefit, therapeutic response is notsatisfactory for the majority of patients treated. It has been estimatedthat significant cognitive improvement occurs in only about 5% oftreated patients and there is little evidence that treatmentsignificantly alters the course of this progressive disease.

Consequently, there remains a tremendous clinical need for moreeffective treatments and in particular those which may arrest or delayprogression of the disease. Also NMDA-receptor antagonists, likememantine, have been employed more recently.

However, adverse events have been reported due to the pharmacologicalactivity. Further, such a treatment with these NMDA-receptor antagonistscan merely be considered as a symptomatic approach and not adisease-modifying one.

Also immunomodulation approaches for the treatment of amyloid-relateddisorders have been proposed. WO 99/27944 discloses conjugates thatcomprise parts of the A-beta peptide and carrier molecules whereby saidcarrier molecule should enhance an immune response. Another activeimmunization approach is mentioned in WO 00/72880, wherein also A-betafragments are employed to induce an immune response.

Also passive immunization approaches with general anti-A-beta antibodieshave been proposed in WO 99/27944 or WO 01/62801 and specific humanizedantibodies directed against portions of A-beta have been described in WO02/46237, WO 02/088306 and WO 02/088307. WO 00/77178 describesantibodies binding a transition state adopted by β-amyloid duringhydrolysis. WO 03/070760 discloses antibody molecules that recognize twodiscontinuous amino acid sequences on the A-beta peptide.

WO 2014/033074 relates to blood brain barrier shuttles that bindreceptors on the blood brain barrier and methods of using the same.Blood-brain barrier drug delivery of IgG fusion proteins with atransferrin receptor monoclonal antibody have been reported byPardridge, W. (Exp. Opin. Drug Deliv. 12 (2015) 207-222). Yu, Y. J. etal. (Sci. Translat. Med. 6 (2014) 261ra154-261ra154) reported thattherapeutic bispecific antibodies cross the blood-brain barrier innonhuman primates. The disaggregation of amyloid plaque in brain ofAlzheimer's disease transgenic mice with daily subcutaneousadministration of a tetravalent bispecific antibody that targets thetransferrin receptor and the abeta amyloid peptide was reported bySumbria, R. K., et al. (Mol. Pharm. 10 (2013) 3507-3513). Niewoehner,J., et al. (Neuron 81 (2014) 49-609 reported an increased brainpenetration and potency of a therapeutic antibody using a monovalentmolecular shuttle.

SUMMARY

Herein is reported a bispecific antibody comprising

-   -   a) one (full length) antibody comprising two pairs each of a        (full length) antibody light chain and a (full length) antibody        heavy chain, wherein the binding sites formed by each of the        pairs of the (full length) heavy chain and the (full length)        light chain specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one of the heavy chains        of the (full length) antibody, wherein the binding site of the        additional Fab fragment specifically binds to a second antigen,    -   wherein each of the (full length) antibody light chains        comprises in the constant light chain domain (CL) at position        123 the amino acid residue arginine (instead of the wild-type        glutamic acid residue; E123R mutation) and at position 124 the        amino acid residue lysine (instead of the wild-type glutamine        residue; Q124K mutation) (numbering according to Kabat),    -   wherein each of the (full length) antibody heavy chains        comprises in the first constant heavy chain domain (CH1) at        position 147 an glutamic acid residue (instead of the wild-type        lysine residue; K147E mutation) and at position 213 an glutamic        acid residue (instead of the wild-type lysine amino acid        residue; K213E mutation) (numbering according to Kabat EU        index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other, and    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor.

In one embodiment the additional Fab fragment is fused to the C-terminusof the heavy chain by a peptidic linker.

In one embodiment the N-terminus of the heavy chain variable domain ofthe Fab fragment is fused to the C-terminus of the full length heavychain or the C-terminus of the peptidic linker.

In one embodiment

-   -   a) the full length heavy chain that is fused to the additional        Fab fragments has as C-terminal heavy chain amino acid residues        the tripeptide LSP wherein the proline thereof is directly fused        to the first amino acid residue of the additional Fab fragment        or of the peptidic linker via a peptide bond, and    -   b) the full length heavy chain that is not fused to the        additional Fab fragments has as C-terminal heavy chain amino        acid residues the tripeptide LSP, or SPG, or PGK.

In one embodiment the (full length) antibody is

-   -   a) a full length antibody of the human subclass IgG1,    -   b) a full length antibody of the human subclass IgG4,    -   c) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G,    -   d) a full length antibody of the human subclass IgG4 with the        mutations S228P, L235E and P329G,    -   e) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G in both heavy chains and the        mutations T366W and S354C in one heavy chain and the mutations        T366S, L368A, Y407V and Y349C in the respective other heavy        chain,    -   f) a full length antibody of the human subclass IgG4 with the        mutations S228P, L235E and P329G in both heavy chains and the        mutations T366W and S354C in one heavy chain and the mutations        T366S, L368A, Y407V and Y349C in the respective other heavy        chain,    -   g) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A, P329G, I253A, H310A and H435A in both        heavy chains and the mutations T366W and S354C in one heavy        chain and the mutations T366S, L368A, Y407V and Y349C in the        respective other heavy chain, or    -   h) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A, P329G, M252Y, S254T and T256E in both        heavy chains and the mutations T366W and S354C in one heavy        chain and the mutations T366S, L368A, Y407V and Y349C in the        respective other heavy chain.

In one embodiment the (full length) antibody is

-   -   a) a full length antibody of the human subclass IgG1,    -   b) a full length antibody of the human subclass IgG4,    -   c) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G,    -   d) a full length antibody of the human subclass IgG4 with the        mutations S228P, L235E and P329G,    -   e) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G in both heavy chains and the        mutations i) T366W, and ii) S354C or Y349C, in one heavy chain        and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or        S354C, in the respective other heavy chain,    -   f) a full length antibody of the human subclass IgG4 with the        mutations S228P, L235E and P329G in both heavy chains and the        mutations i) T366W, and ii) S354C or Y349C, in one heavy chain        and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or        S354C, in the respective other heavy chain,    -   g) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A, P329G, I253A, H310A and H435A in both        heavy chains and the mutations i) T366W, and ii) S354C or Y349C,        in one heavy chain and the mutations i) T366S, L368A, and Y407V,        and ii) Y349C or S354C, in the respective other heavy chain,    -   h) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A, P329G, M252Y, S254T and T256E in both        heavy chains and the mutations i) T366W, and ii) S354C or Y349C,        in one heavy chain and the mutations i) T366S, L368A, and Y407V,        and ii) Y349C or S354C, in the respective other heavy chain, or    -   i) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A, P329G, H310A, H433A and Y436A in both        heavy chains and the mutations i) T366W, and ii) S354C or Y349C,        in one heavy chain and the mutations i) T366S, L368A, and Y407V,        and ii) Y349C or S354C, in the respective other heavy chain.

In one embodiment the additional Fab fragment is fused to the C-terminusof the heavy chain comprising the mutation T366W, or to the C-terminusof the heavy chain comprising the mutations T366S, L368A, and Y407V.

In one embodiment

-   -   the full length antibody is of the human subclass IgG1 with the        mutations L234A, L235A and P329G in both heavy chains and the        mutations T366W and S354C in one heavy chain and the mutations        T366S, L368A, Y407V and Y349C in the respective other heavy        chain, and    -   the additional Fab fragment is fused to the C-terminus of the        heavy chain comprising the mutation T366W, or to the C-terminus        of the heavy chain comprising the mutations T366S, L368A, and        Y407V.

In one embodiment of all aspects, the human A-beta binding sitecomprises the VH sequence as in SEQ ID NO: 18, includingpost-translational modifications of that sequence, and the VL sequenceas in SEQ ID NO: 19, including post-translational modifications of thatsequence.

In one embodiment of all aspects, the human transferrin receptor bindingsite comprises the VH sequence as in SEQ ID NO: 20, includingpost-translational modifications of that sequence, and the VL sequenceas in SEQ ID NO: 21, including post-translational modifications of thatsequence.

In one embodiment the bispecific antibody comprises

-   -   i) a light chain with an amino acid sequence that has a sequence        identity to SEQ ID NO: 01 of 70% or more,    -   ii) a heavy chain with an amino acid sequence that has a        sequence identity to SEQ ID NO: 02 of 70% or more,    -   iii) a light chain with an amino acid sequence that has a        sequence identity to SEQ ID NO: 03 of 70% or more, and    -   iv) a heavy chain Fab fragment with an amino acid sequence that        has a sequence identity to SEQ ID NO: 04 of 70% or more,    -   wherein    -   SEQ ID NO: 01 has the amino acid sequence

DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC,

-   -   SEQ ID NO: 02 has the amino acid sequence

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

-   -   SEQ ID NO: 03 has the amino acid sequence

AIQLTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYASSNVDNTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSC,and

-   -   SEQ ID NO: 04 has the amino acid sequence

QSMQESGPGLVKPSQTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISKTSTTVSLKLSSVTAADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

One aspect as reported herein is a bispecific antibody comprising a(full length) light chain that has the amino acid sequence of SEQ ID NO:01, a (full length) heavy chain that has the amino acid sequence of SEQID NO: 02, a (full length) light chain that has the amino acid sequenceof SEQ ID NO: 03, and an antibody Fab fragment comprising the amino acidsequences of SEQ ID NO: 04.

In one embodiment the bispecific antibody is monoclonal.

One aspect as reported herein is an isolated nucleic acid encoding thebispecific antibody as reported herein.

One aspect as reported herein is a host cell comprising the nucleic acidas reported herein encoding the bispecific antibody as reported herein.

One aspect as reported herein is a method of producing a bispecificantibody as reported herein comprising the following steps:

-   -   a) culturing the host cell as reported herein so that the        bispecific antibody is produced, and    -   b) recovering the bispecific antibody from the cell or the        cultivation medium and thereby producing the bispecific antibody        as reported herein.

One aspect as reported herein is an immunoconjugate comprising thebispecific antibody as reported herein and a cytotoxic agent.

One aspect as reported herein is a pharmaceutical formulation comprisingthe bispecific antibody as reported herein and a pharmaceuticallyacceptable carrier.

One aspect as reported herein is the antibody as reported herein for useas a medicament.

One aspect as reported herein is the bispecific antibody as reportedherein for use in treating Alzheimer's disease.

One aspect as reported herein is the bispecific antibody as reportedherein for use in inhibiting/slowing down the formation of plaques inthe brain.

One aspect as reported herein is the bispecific antibody as reportedherein for use in disintegrating β-amyloid plaques.

One aspect as reported herein is the use of the bispecific antibody asreported herein in the manufacture of a medicament.

In one embodiment the medicament is for the treatment of amyloiddisorders.

In one embodiment the medicament for the prevention and/or treatment ofa disease associated with amyloidogenesis and/or amyloid-plaqueformation. In one embodiment the disease is selected from the groupconsisting of dementia, Alzheimer's disease, motor neuropathy, Down'ssyndrome, Creutzfeldt Jacob disease, hereditary cerebral hemorrhage withamyloidosis Dutch type, Parkinson's disease, HIV-related dementia, ALSor neuronal disorders related to aging. In one preferred embodiment themedicament is for treatment of Alzheimer's disease.

In one embodiment the medicament is for inhibiting/slowing down theformation of plaques in the brain. In one embodiment the medicament isfor medicament for the disintegration of β-amyloid plaques.

One aspect as reported herein is a method of treating an individualhaving a disease associated with amyloidogenesis and/or amyloid-plaqueformation comprising administering to the individual an effective amountof the bispecific antibody as reported herein.

One aspect as reported herein is a method of treating an individualhaving Alzheimer's disease comprising administering to the individual aneffective amount of the bispecific antibody as reported herein.

One aspect as reported herein is a method for the disintegration ofβ-amyloid plaques in the brain of an individual comprising administeringto the individual an effective amount of the bispecific antibody asreported herein to disintegrate β-amyloid plaques in the brain.

One aspect as reported herein is a method of inhibiting/slowing down theformation of plaques in the brain of an individual comprisingadministering to the individual an effective amount of the bispecificantibody as reported herein to inhibit/slow down the formation ofplaques in the brain.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The knobs into holes dimerization modules and their use in antibodyengineering are described in Carter P.; Ridgway J. B. B.; Presta L. G.:Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73. Theadditional disulfide bridge in the CH3 domain is reported in Merchant,A. M., et al., Nat. Biotechnol. 16 (1998) 677-681.

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and is referred to as“numbering according to Kabat” herein. Specifically, the Kabat numberingsystem (see pages 647-660) of Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, MD (1991) is used for the light chainconstant domain CL of kappa and lambda isotype, and the Kabat EU indexnumbering system (see pages 661-723) is used for the constant heavychain domains (CH1, Hinge, CH2 and CH3, which is herein furtherclarified by referring to “numbering according to Kabat EU index” inthis case).

I. Definitions

The “blood-brain-barrier” or “BBB” refers to the physiological barrierbetween the peripheral circulation and the brain and spinal cord whichis formed by tight junctions within the brain capillary endothelialplasma membranes, creating a tight barrier that restricts the transportof molecules into the brain, even very small molecules such as urea (60Daltons). The BBB within the brain, the blood-spinal-cord barrier withinthe spinal cord, and the blood-retinal-barrier within the retina arecontiguous capillary barriers within the CNS, and are hereincollectively referred to an the blood-brain-barrier or BBB. The BBB alsoencompasses the blood-CSF barrier (choroid plexus) where the barrier iscomprised of ependymal cells rather than capillary endothelial cells.

The terms “anti-human A-beta antibody” and “an antibody specificallybinding to human A-beta” refer to an antibody that is capable of bindingthe human A-beta peptide with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting A-betapeptide.

It is of note that human A-beta has several naturally occurring forms,whereby the human forms are referred to as Aβ39, Aβ40, Aβ41, Aβ42 andAβ43. The most prominent form, Aβ42, has the amino acid sequence of SEQID NO: 05. In Aβ41, Aβ40, Aβ39, the C-terminal amino acids A, IA and VIAare missing, respectively. In the Aβ43 form an additional threonineresidue is comprised at the C-terminus of SEQ ID NO: 05. In oneembodiment the human A-beta protein has the amino acid sequence of SEQID NO: 05.

Thus, the term also encompasses antibodies that bind to a shortenedfragment of the human A-beta polypeptide.

The “central nervous system” or “CNS” refers to the complex of nervetissues that control bodily function, and includes the brain and spinalcord.

A “blood-brain-barrier receptor” (abbreviated “BBBR” herein) is anextracellular membrane-linked receptor protein expressed on brainendothelial cells which is capable of transporting molecules across theBBB or be used to transport exogenous administrated molecules. Examplesof BBBR herein include: transferrin receptor (TfR), insulin receptor,insulin-like growth factor receptor (IGF-R), low density lipoproteinreceptors including without limitation low density lipoproteinreceptor-related protein 1 (LRP1) and low density lipoproteinreceptor-related protein 8 (LRP8), and heparin-binding epidermal growthfactor-like growth factor (HB-EGF). One preferred BBBR is transferrinreceptor (TfR).

The “transferrin receptor” (“TfR”) is a transmembrane glycoprotein (witha molecular weight of about 180,000 Da) composed of twodisulphide-bonded sub-units (each of apparent molecular weight of about90,000 Da) involved in iron uptake in vertebrates. In one embodiment,the TfR as mentioned herein is human TfR comprising the amino acidsequence as in Schneider et al (Nature 311 (1984) 675-678), for example.In one embodiment the human transferrin receptor has the amino acidsequence of SEQ ID NO: 22.

A “multispecific antibody” denotes an antibody having bindingspecificities for at least two different epitopes on the same antigen ortwo different antigens. Exemplary multispecific antibodies may bind botha BBBR and a brain antigen. Multispecific antibodies can be prepared asfull-length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies) or combinations thereof (e.g. full length antibody plusadditional scFv or Fab fragments). Engineered antibodies with two, threeor more (e.g. four) functional antigen binding sites have also beenreported (see, e.g., US 2002/0004587 A1).

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (k_(d)). Affinity can be measured by common methods known inthe art, including those described herein.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies) so long as they exhibit the desiredantigen-binding activity.

The term “antibody-dependent cellular cytotoxicity (ADCC)” is a functionmediated by Fc receptor binding and refers to lysis of target cells byan antibody as reported herein in the presence of effector cells. ADCCis measured in one embodiment by the treatment of a preparation of CD19expressing erythroid cells (e.g. K562 cells expressing recombinant humanCD19) with an antibody as reported herein in the presence of effectorcells such as freshly isolated PBMC (peripheral blood mononuclear cells)or purified effector cells from buffy coats, like monocytes or NK(natural killer) cells. Target cells are labeled with ⁵¹Cr andsubsequently incubated with the antibody. The labeled cells areincubated with effector cells and the supernatant is analyzed forreleased ⁵¹Cr. Controls include the incubation of the target endothelialcells with effector cells but without the antibody. The capacity of theantibody to induce the initial steps mediating ADCC is investigated bymeasuring their binding to Fcγ receptors expressing cells, such ascells, recombinantly expressing FcγRI and/or FcγRIIA or NK cells(expressing essentially FcγRIIIA). In one preferred embodiment bindingto FcγR on NK cells is measured.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

The term “complement-dependent cytotoxicity (CDC)” refers to lysis ofcells induced by the antibody as reported herein in the presence ofcomplement. CDC is measured in one embodiment by the treatment of CD19expressing human endothelial cells with an antibody as reported hereinin the presence of complement.

The cells are in one embodiment labeled with calcein. CDC is found ifthe antibody induces lysis of 20% or more of the target cells at aconcentration of 30 μg/ml. Binding to the complement factor C1q can bemeasured in an ELISA. In such an assay in principle an ELISA plate iscoated with concentration ranges of the antibody, to which purifiedhuman C1q or human serum is added. C1q binding is detected by anantibody directed against C1q followed by a peroxidase-labeledconjugate. Detection of binding (maximal binding Bmax) is measured asoptical density at 405 nm (OD405) for peroxidase substrate ABTS®(2,2′-azino-di-[3-ethylbenzthiazoline-6-sulfonate (6)]).

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody class.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

Fc receptor binding dependent effector functions can be mediated by theinteraction of the Fc-region of an antibody with Fc receptors (FcRs),which are specialized cell surface receptors on hematopoietic cells. Fcreceptors belong to the immunoglobulin superfamily, and have been shownto mediate both the removal of antibody-coated pathogens by phagocytosisof immune complexes, and the lysis of erythrocytes and various othercellular targets (e.g. tumor cells) coated with the correspondingantibody, via antibody dependent cell mediated cytotoxicity (ADCC) (seee.g. Van de Winkel, J. G. and Anderson, C. L., J. Leukoc. Biol. 49(1991) 511-524). FcRs are defined by their specificity forimmunoglobulin isotypes: Fc receptors for IgG antibodies are referred toas FcγR. Fc receptor binding is described e.g. in Ravetch, J. V. andKinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., etal., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin.Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76(1998) 231-248.

Cross-linking of receptors for the Fc-region of IgG antibodies (FcγR)triggers a wide variety of effector functions including phagocytosis,antibody-dependent cellular cytotoxicity, and release of inflammatorymediators, as well as immune complex clearance and regulation ofantibody production. In humans, three classes of FcγR have beencharacterized, which are:

-   -   FcγRI (CD64) binds monomeric IgG with high affinity and is        expressed on macrophages, monocytes, neutrophils and        eosinophils. Modification in the Fc-region IgG at least at one        of the amino acid residues E233-G236, P238, D265, N297, A327 and        P329 (numbering according to EU index of Kabat) reduce binding        to FcγRI. IgG2 residues at positions 233-236, substituted into        IgG1 and IgG4, reduced binding to FcγRI by 10³-fold and        eliminated the human monocyte response to antibody-sensitized        red blood cells (Armour, K. L., et al., Eur. J. Immunol.        29 (1999) 2613-2624).    -   FcγRII (CD32) binds complexed IgG with medium to low affinity        and is widely expressed. This receptor can be divided into two        sub-types, FcγRIIA and FcγRIIB. FcγRIIA is found on many cells        involved in killing (e.g. macrophages, monocytes, neutrophils)        and seems able to activate the killing process. FcγRIIB seems to        play a role in inhibitory processes and is found on B-cells,        macrophages and on mast cells and eosinophils. On B-cells it        seems to function to suppress further immunoglobulin production        and isotype switching to, for example, the IgE class. On        macrophages, FcγRIIB acts to inhibit phagocytosis as mediated        through FcγRIIA. On eosinophils and mast cells the B-form may        help to suppress activation of these cells through IgE binding        to its separate receptor. Reduced binding for FcγRIIA is found        e.g. for antibodies comprising an IgG Fc-region with mutations        at least at one of the amino acid residues E233-G236, P238,        D265, N297, A327, P329, D270, Q295, A327, R292, and K414        (numbering according to EU index of Kabat).    -   FcγRIII (CD16) binds IgG with medium to low affinity and exists        as two types. FcγRIIIA is found on NK cells, macrophages,        eosinophils and some monocytes and T cells and mediates ADCC.        FcγRIIIB is highly expressed on neutrophils. Reduced binding to        FcγRIIIA is found e.g. for antibodies comprising an IgG        Fc-region with mutation at least at one of the amino acid        residues E233-G236, P238, D265, N297, A327, P329, D270, Q295,        A327, 5239, E269, E293, Y296, V303, A327, K338 and D376        (numbering according to EU index of Kabat).

Mapping of the binding sites on human IgG1 for Fc receptors, the abovementioned mutation sites and methods for measuring binding to FcγRI andFcγRIIA are described in Shields, R. L., et al. J. Biol. Chem. 276(2001) 6591-6604.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc receptor” as used herein refers to activation receptorscharacterized by the presence of a cytoplasmatic ITAM sequenceassociated with the receptor (see e.g. Ravetch, J. V. and Bolland, S.,Annu. Rev. Immunol. 19 (2001) 275-290). Such receptors are FcγRI,FcγRIIA and FcγRIIIA. The term “no binding of FcγR” denotes that at anantibody concentration of 10 μg/ml the binding of an antibody asreported herein to NK cells is 10% or less of the binding found foranti-OX40L antibody LC.001 as reported in WO 2006/029879.

While IgG4 shows reduced FcR binding, antibodies of other IgG subclassesshow strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fccarbohydrate), Pro329 and 234, 235, 236 and 237 Ile253, Ser254, Lys288 ,Thr307, Gln311, Asn434, and His435 are residues which provide if alteredalso reduce FcR binding (Shields, R.L., et al. J. Biol. Chem. 276 (2001)6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan, A., etal., Immunology 86 (1995) 319-324; and EP 0 307 434). In one embodimentthe antibody as reported herein is of IgG1 or IgG2 subclass andcomprises the mutation PVA236, GLPSS331, and/or L234A/L235A. In oneembodiment the antibody as reported herein is of IgG4 subclass andcomprises the mutation L235E. In one embodiment the antibody furthercomprises the mutation S228P.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication 91-3242.

The antibodies as reported herein comprise as Fc-region, in oneembodiment an Fc-region derived from human origin. In one embodiment theFc-region comprises all parts of the human constant region. TheFc-region of an antibody is directly involved in complement activation,C1q binding, C3 activation and Fc receptor binding. While the influenceof an antibody on the complement system is dependent on certainconditions, binding to C1q is caused by defined binding sites in theFc-region. Such binding sites are known in the state of the art anddescribed e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16 (1979)907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E. E., et al.,J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75(2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324;and EP 0 307 434. Such binding sites are e.g. L234, L235, D270, N297,E318, K320, K322, P331 and P329 (numbering according to EU index ofKabat; Unless otherwise specified herein, numbering of amino acidresidues in the Fc-region or constant region is according to the EUnumbering system, also called the EU index, as described in Kabat, E. A.et al., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991), NIHPublication 91-3242). Antibodies of subclass IgG1, IgG2 and IgG3 usuallyshow complement activation, C1q binding and C3 activation, whereas IgG4do not activate the complement system, do not bind C1q and do notactivate C3. An “Fc-region of an antibody” is a term well known to theskilled artisan and defined on the basis of papain cleavage ofantibodies. In one embodiment the Fc-region is a human Fc-region. In oneembodiment the Fc-region is of the human IgG4 subclass comprising themutations S228P and/or L235E (numbering according to EU index of Kabat).In one embodiment the Fc-region is of the human IgG1 subclass comprisingthe mutations L234A and L235A (numbering according to EU index ofKabat).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody”, “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein. A “fulllength antibody” is an antibody that comprises a light and heavy chainantigen-binding variable region (VL, VH) as well as a light chainconstant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.The constant domains may be native sequence constant domains (e.g. humannative sequence constant domains) or amino acid sequence variantsthereof. In more detail a full length antibody comprises two antibodylight chains (each comprising a light chain variable domain and a lightchain constant domain) and two antibody heavy chains (each comprising aheavy chain variable domain, a hinge region and the heavy chain constantdomains CH1, CH2 and CH3). The C-terminal amino acid residues K or GKmay be present or not independently of each other in the two antibodyheavy chains of a full length antibody. Also a full length antibody maycomprise amino acid additions, mutations and deletions within thedomains but not the deletion of an entire domain.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Bethesda Md. (1991), NIH Publication 91-3242, Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain comprising the amino acidresidue stretches which are hypervariable in sequence (“complementaritydetermining regions” or “CDRs”) and/or form structurally defined loops(“hypervariable loops”), and/or contain the antigen-contacting residues(“antigen contacts”). Generally, antibodies comprise six HVRs; three inthe VH (H1, H2, H3), and three in the VL (L1, L2, L3).

HVRs include

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987)        901-917);    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat, E. A. et al., Sequences of Proteins of Immunological        Interest, 5th ed. Public Health Service, National Institutes of        Health, Bethesda, Md. (1991), NIH Publication 91-3242.);    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including amino acid        residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35        (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-human A-beta/human transferrinreceptor antibody” refers to one or more nucleic acid molecules encodingantibody heavy and light chains (or fragments thereof), including suchnucleic acid molecule(s) in a single vector or separate vectors, andsuch nucleic acid molecule(s) present at one or more locations in a hostcell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),whereby between the first and the second constant domain a hinge regionis located. Similarly, from N- to C-terminus, each light chain has avariable region (VL), also called a variable light domain or a lightchain variable domain, followed by a constant light (CL) domain. Thelight chain of an antibody may be assigned to one of two types, calledkappa (κ) and lambda (λ), based on the amino acid sequence of itsconstant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject., A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies asreported herein are used to delay development of a disease or to slowthe progression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt,T. J. et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., N.Y.(2007), page 91) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano, S. et al., J.Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991)624-628).

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

II. Compositions and Methods

In one aspect, the invention is based, in part, on the finding that thebispecific anti-human A-beta/human transferrin receptor antibody asreported herein has improved properties. In certain embodiments,bispecific anti-human A-beta/human transferrin receptor antibodies areprovided. Antibodies as reported herein are useful, e.g., for thediagnosis or treatment of Alzheimer's disease.

A. Exemplary Bispecific Anti-Human A-Beta/Human Transferrin ReceptorAntibodies

In one aspect, the invention provides isolated bispecific antibodiesthat bind to human A-beta and human transferrin receptor. The antibodiesare bispecific antibodies consisting of a full length core antibody anda fused Fab fragment in which certain domains are crosswise exchanged.Thus, the resulting bispecific antibody is asymmetric. Therefore, thebispecific antibody is produced using the heterodimerization technologycalled knobs-into-holes using a first heavy chain with the so-calledknob mutations (HCknob) and a second heavy chain with the so-called holemutations (HChole).

Antibody 0012, which is also an aspect of the current invention, iscomposed of four polypeptides that have the amino acid sequence of SEQID NO: 06 to 09.

Antibody 0012 is a bispecific antibody comprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the light        chain variable domain (VL) and the heavy chain variable domain        (VH) are replaced by each other, and    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor.

Antibody 0015, which is also an aspect of the current invention, iscomposed of four polypeptides that have the amino acid sequence of SEQID NO: 01 to 03 and SEQ ID NO: 10.

Antibody 0015 is a bispecific antibody comprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other, and wherein the first        antigen is human A-beta protein and the second antigen is human        transferrin receptor.

Antibody 0020, which is also an aspect of the current invention, iscomposed of three polypeptides that have the amino acid sequence of SEQID NO: 11 to 13.

Antibody 0020 is a bispecific antibody comprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen is a single chain Fab fragment, and    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor.

Antibody 0024, which is also an aspect of the current invention, iscomposed of four polypeptides that have the amino acid sequence of SEQID NO: 14 to 17.

Antibody 0024 is a bispecific antibody comprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other, and    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor.

Different allocation/combination of the respective polypeptides ondifferent expression plasmids and different ratios of the resultingplasmids have been used for the recombinant production of the bispecificantibodies. The results are presented in the following Table.

relative peak area [%] (CE-SDS; non-reduced analysis) hole- ½ hole mAbchain antibody antibody plasmid ratio LC hole dimer monomer 00121(HChole + LC):3(HCknob + 12 9 78 LCcross) 0012 l(LC):1(HChole + LC): 19 9 79 3(HCknob + LCcross) 0012 1(LC + HChole):3(Hcknob + 6 9 9 75LC):1(LCcross) 0015 1(HChole + LC):3(HCknob + 7 23 62 LCcross) 0015l(LC):1(HChole + LC): 4 17 75 3(HCknob + LCcross) 0015 1(LC +HChole):3(Hcknob + 4 20 66 LC):1(LCcross) 0020 1(HChole + LC):4(HCknob +16 11 72 LCcross)

The bispecific antibodies have been produced in small scale and theby-product distribution has been analyzed after a first purificationstep using a protein A affinity chromatography and after the secondpurification step using a preparative size-exclusion chromatography. Theresults are presented in the following Table.

harvest 3 liter by-product distribution fermentation (CE-SDS not red.)after preparative hole- protein A hole product monomer ½ dimer + plasmid(CE-SDS not mAb ¾ antibody ratio red./yield) LC hole mAb 0012 1:1:3 65% 3% 28% 35% 13.3 mg 0024 1:1:3 70%  6% 15%  7% 14.8 mg 0015 1:1:3 85% 4%  5%  5% 15.8 mg 0020 1:4 29% 11% 44%  8%    6 mg

harvest 3 liter fermentation by-product distribution after preparative(CE-SDS not red.) protein A and hole- preparative SEC hole productmonomer ½ dimer + plasmid (CE-SDS not mAb ¾ antibody ratio red./yield)LC hole mAb 0012 1:1:3 >90%  5%   3% 2.5% 2.8 mg   0024 1:1:3   78% 11%  5%   6%   4 mg 0015 1:1:3 >95%  1% 0.5%   1% 5.8 mg 0020 1:4   68% 13% 10% 8.6% 0.8 mg

harvest 31 after preparative protein A plas- purification by-productsend by-products anti- mid monomer SEC [%] product SEC [%] body ratio bySEC HMW LMW by SEC HMW LMW 0012 1:1:3 78% 0 22 97.5% 0 2.5 0024 1:1:380% 0 20   96% 0 4   0015 1:1:3 87% 0 13   97% 0 3   0020 1:4   53% 7 40  97% 0 3  

The anti-A-beta binding site comprises an additional glycosylation site.Therefore the glycosylation has been determined. The results arepresented in the following Table.

HChole HCknob CE-SDS; reduced; rel. peak area [%] non- non- antibodyplasmid ratio glyc. glyc. glyc. glyc. 0012 1(HChole + LC): 10 14 8 183(HCknob + LCcross) 0012 1(LC):1(HChole + LC): 9 13 8 21 3(HCknob +LCcross) 0012 1(LC + HChole): 10 13 7 18 3(Hcknob + LC): 1(LCcross) 00151(HChole + LC): 7 16 5 25 3(HCknob + LCcross) 0015 1(LC):1(HChole + LC):6 16 5 29 3(HCknob + LCcross) 0015 1(LC + HChole): 7 15 6 26 3(Hcknob +LC): 1(LCcross) 0020 1(HChole + LC): 9 18 7 21 4(HCknob + LCcross)

Percentage of non-glycosylated Fab in 3 liter fermentations after ProtApurification

HChole HCknob CE-SDS; reduced non- non- antibody plasmid ratio glyc.glyc. glyc. glyc. 0012 1(LC):1(HChole + LC): 23 77 10 90 3(HCknob +LCcross) 0015 1(LC):1(HChole + LC): 16 84 8 92 3(HCknob + LCcross) 00241(LC):1(HChole + LC): 16 84 7.5 92.5 3(HCknob + LCcross)

Percentage of non-glycosylated Fab in 31 fermentations after ProtA+SECpurification

HChole HCknob CE-SDS; reduced non- non- antibody plasmid ratio glyc.glyc. glyc. glyc. 0012 1(LC):1(HChole + LC): 8 92 6 94 3(HCknob +LCcross) 0015 1(LC):1(HChole + LC): 10 90 4.5 95.5 3(HCknob + LCcross)0024 1(LC):1(HChole + LC): 8 92 4.5 95.5 3(HCknob + LCcross)

The stability of the bispecific antibodies has been tested by incubationfor 14 day at specific pH values in buffer. The results are presented inthe following Table.

antibody antibody antibody parameter 0012 0015 0024 relative A- 14 days,pH  87% 96%  97% beta peptide 6.0, 40° C., (1-40) binding His/NaCl byBIAcore buffer 14 days, pH  82% 86% 101% 7.4, 37° C., PBS bufferrelative 14 days, pH 101% 91%  93% human 6.0, 40° C., transferrinHis/NaCl receptor buffer binding by 14 days, pH  78% 85%  90% BIAcore7.4, 37° C., PBS buffer

The aggregation temperature for antibody 0015 and 0024 was determined tobe approx. 53-55° C. and for antibody 0012 to be approx. 54-56° C.

The off-rate (k_(d) in [1/Ms]) for the binding to the human transferrinreceptor as determined by BIAcore was comparable for antibodies 0015 and0024 as well as for the parental anti-human transferrin receptorantibody at 25° C., 37° C. and 40° C.: 1.86E-02 to 1.97E-02, 1.98E-2 to2.03E-2 and 1.44E-02, respectively.

The A-beta specific effector function of all antibodies were comparableto the parental monospecific anti-A-beta antibody in a U937-cell assay.The data is presented in the following Table.

antibody IL-8 [ng/ml] IP-10 [ng/ml] parental anti-A-beta 5 4.3 antibodyantibody-0012 7 5.3 antibody-0015 — 5 antibody-0020 7 4.5 antibody-00248 5.2

None of the bispecific antibodies showed neutrophil activation in vitro.

Overall antibody 0015 showed suitable properties and is therefore thepreferred aspect of the invention. Furthermore this antibody hasimproved properties, which lie, amongst others, in the improvedside-product profile.

In one aspect herein is provided a bispecific antibody comprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other, and wherein the first        antigen is human A-beta protein and the second antigen is human        transferrin receptor.

Another aspect as reported herein is a bispecific antibody comprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other,    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor,    -   wherein the human A-beta binding site comprises a heavy chain        variable domain (VH) sequence having at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to        the amino acid sequence of SEQ ID NO: 18 and a light chain        variable domain (VL) sequence having at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to        the amino acid sequence of SEQ ID NO: 19, and    -   wherein the human transferrin receptor binding site comprises a        heavy chain variable domain (VH) sequence having at least 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence        identity to the amino acid sequence of SEQ ID NO: 20 and a light        chain variable domain (VL) sequence having at least 90%, 91%,        92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence        identity to the amino acid sequence of SEQ ID NO: 21.

In certain embodiments, a VH sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but a binding site comprising that sequenceretains the ability to bind to its antigen. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 18 or 20. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs).

In certain embodiments, a VL sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but a binding site comprising that sequenceretains the ability to bind to its antigen. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 19 or 21. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs).

In one embodiment, the human A-beta binding site comprises the VHsequence as in SEQ ID NO: 18, including post-translational modificationsof that sequence, and the VL sequence as in SEQ ID NO: 19, includingpost-translational modifications of that sequence.

In one embodiment, the human transferrin receptor binding site comprisesthe VH sequence as in SEQ ID NO: 20, including post-translationalmodifications of that sequence, and the VL sequence as in SEQ ID NO: 21,including post-translational modifications of that sequence.

In one embodiment the bispecific antibody comprises

-   -   i) a light chain that has a sequence identity to SEQ ID NO: 01        of 70-100%, at least 70%, at least 80%, at least 90%, or 95% or        more,    -   ii) a heavy chain that has a sequence identity to SEQ ID NO: 02        of 70-100%, at least 70%, at least 80%, at least 90%, or 95% or        more,    -   iii) a light chain that has a sequence identity to SEQ ID NO: 03        of 70-100%, at least 70%, at least 80%, at least 90%, or 95% or        more, and    -   iv) a heavy chain Fab fragment that has a sequence identity to        SEQ ID NO: 04 of 70-100%, at least 70%, at least 80%, at least        90%, or 95% or more,    -   wherein    -   SEQ ID NO: 01 has the amino acid sequence

DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC,

-   -   SEQ ID NO: 02 has the amino acid sequence

QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

-   -   SEQ ID NO: 03 has the amino acid sequence

AIQLTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYASSNVDNTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSC,and

-   -   SEQ ID NO: 04 has the amino acid sequence

QSMQESGPGLVKPSQTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISKTSTTVSLKLSSVTAADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Another aspect as reported herein is a bispecific antibody comprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other, wherein the first        antigen is human A-beta protein and the second antigen is human        transferrin receptor,    -   wherein the human A-beta binding site comprises a heavy chain        variable domain (VH) that has the amino acid sequence of SEQ ID        NO: 18 and a light chain variable domain (VL) that has the amino        acid sequence of SEQ ID NO: 19, and    -   wherein the human transferrin receptor binding site comprises a        heavy chain variable domain (VH) that has the amino acid        sequence of SEQ ID NO: 20 and a light chain variable domain (VL)        that has the amino acid sequence of SEQ ID NO: 21.

Another aspect as reported herein is a bispecific antibody comprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, wherein the full length        antibody comprises an Fc-region that is formed by the Fc-region        polypeptides, each comprising the CH1, CH2 and CH3 domain, of        the two full length heavy chains, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other,    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor,    -   wherein the human A-beta binding site comprises a heavy chain        variable domain (VH) that has the amino acid sequence of SEQ ID        NO: 18 and a light chain variable domain (VL) that has the amino        acid sequence of SEQ ID NO: 19,    -   wherein the human transferrin receptor binding site comprises a        heavy chain variable domain (VH) that has the amino acid        sequence of SEQ ID NO: 20 and a light chain variable domain (VL)        that has the amino acid sequence of SEQ ID NO: 21, and    -   wherein the Fc-region polypeptides are    -   a) of the human subclass IgG1,    -   b) of the human subclass IgG4,    -   c) of the human subclass IgG1 with the mutations L234A, L235A        and P329G,    -   d) of the human subclass IgG4 with the mutations S228P, L235E        and P329G,    -   e) of the human subclass IgG1 with the mutations L234A, L235A        and P329G in both Fc-region polypeptides and the mutations T366W        and S354C in one Fc-region polypeptide and the mutations T366S,        L368A, Y407V and Y349C in the respective other Fc-region        polypeptide,    -   f) of the human subclass IgG4 with the mutations S228P and P329G        in both Fc-region polypeptides and the mutations T366W and S354C        in one Fc-region polypeptide and the mutations T366S, L368A,        Y407V and Y349C in the respective other Fc-region polypeptide,    -   g) of the human subclass IgG1 with the mutations L234A, L235A,        P329G, I253A, H310A and H435A in both Fc-region polypeptides and        the mutations T366W and S354C in one Fc-region polypeptide and        the mutations T366S, L368A, Y407V and Y349C in the respective        other Fc-region polypeptide,    -   h) of the human subclass IgG1 with the mutations L234A, L235A,        P329G, M252Y, S254T and T256E in both Fc-region polypeptides and        the mutations T366W and S354C in one Fc-region polypeptide and        the mutations T366S, L368A, Y407V and Y349C in the respective        other Fc-region polypeptide, or    -   i) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A, P329G, H310A, H433A and Y436A in both        heavy chains and the mutations i) T366W, and ii) S354C or Y349C,        in one heavy chain and the mutations i) T366S, L368A, and Y407V,        and ii) Y349C or S354C, in the respective other heavy chain.

In a further aspect, a bispecific anti-human A-beta/human transferrinreceptor antibody according to any of the above embodiments mayincorporate any of the features, singly or in combination, as describedin Sections 1-3 below:

1. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I. et al., Nature 332 (1988)323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S.V. et al., Methods 36 (2005) 25-34 (describingspecificity determining region (SDR) grafting); Padlan, E. A., Mol.Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, W. F.et al., Methods 36 (2005) 43-60 (describing “FR shuffling”); andOsbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br.J. Cancer 83 (2000) 252-260 (describing the “guided selection” approachto FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J. et al., J. Immunol. 151 (1993) 2296-2308;framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89(1992) 4285-4289; and Presta, L. G. et al., J. Immunol. 151 (1993)2623-2632); human mature (somatically mutated) framework regions orhuman germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

2. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk, M. A. and vande Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg,N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and US 2007/0061900, describing VELOCIMOUSE® technology). Human variableregions from intact antibodies generated by such animals may be furthermodified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor, D.,J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147(1991) 86-95) Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J. et al., Proc. Natl. Acad. Sci.USA 103 (2006) 35.57-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines) and Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

3. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in the Table below under the heading of “preferred substitutions”.More substantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, P. S.,Methods Mol. Biol. 207 (2008) 179-196), and/or residues that contactantigen, with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described, e.g., in Hoogenboom, H. R. etal. in Methods in Molecular Biology 178 (2002) 1-37. In some embodimentsof affinity maturation, diversity is introduced into the variable geneschosen for maturation by any of a variety of methods (e.g., error-pronePCR, chain shuffling, or oligonucleotide-directed mutagenesis). Asecondary library is then created. The library is then screened toidentify any antibody variants with the desired affinity. Another methodto introduce diversity involves HVR-directed approaches, in whichseveral HVR residues (e.g., 4-6 residues at a time) are randomized. HVRresidues involved in antigen binding may be specifically identified,e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham, B. C. and Wells, J. A., Science244 (1989) 1081-1085. In this method, a residue or group of targetresidues (e.g., charged residues such as arg, asp, his, lys, and glu)are identified and replaced by a neutral or negatively charged aminoacid (e.g., alanine or polyalanine) to determine whether the interactionof the antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright, A. and Morrison, S. L., TIBTECH 15 (1997)26-32. The oligosaccharide may include various carbohydrates, e.g.,mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, aswell as a fucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody as reported herein may be made in orderto create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US 2003/0157108; US 2004/0093621. Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A.et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al.,Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable ofproducing defucosylated antibodies include Lec13 CHO cells deficient inprotein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249(1986) 533-545; US 2003/0157108; and WO 2004/056312, especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y.et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, herein is contemplated an antibody variant thatpossesses some but not all effector functions, which make it a desirablecandidate for applications in which the half-life of the antibody invivo is important yet certain effector functions (such as complement andADCC) are unnecessary or deleterious. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express Fc(RIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch, J.V. and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492. Non-limitingexamples of in vitro assays to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom,I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; andHellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166(1987) 1351-1361). Alternatively, non-radioactive assays methods may beemployed (see, for example, ACTI™ non-radioactive cytotoxicity assay forflow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, Wisc.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes, R.et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assaysmay also be carried out to confirm that the antibody is unable to bindC1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISAin WO 2006/029879 and WO 2005/100402. To assess complement activation, aCDC assay may be performed (see, for example, Gazzano-Santoro, H. etal., J. Immunol. Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood101 (2003) 1045-1052; and Cragg, M. S. and M. J. Glennie, Blood 103(2004) 2738-2743). FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int. Immunol. 18 (2006: 1759-1769).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164(2000) 4178-4184.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No.7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding a bispecific anti-human A-beta/humantransferrin receptor antibody described herein is provided. Such nucleicacid may encode an amino acid sequence comprising the VL and/or an aminoacid sequence comprising the VH of the antibody (e.g., the light and/orheavy chains of the antibody). In a further embodiment, one or morevectors (e.g., expression vectors) comprising such nucleic acid areprovided. In a further embodiment, a host cell comprising such nucleicacid is provided. In one such embodiment, a host cell comprises (e.g.,has been transformed with): (1) a vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and anamino acid sequence comprising the VH of the antibody, or (2) a firstvector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and a second vector comprising anucleic acid that encodes an amino acid sequence comprising the VH ofthe antibody. In one embodiment, the host cell is eukaryotic, e.g. aChinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20cell). In one embodiment, a method of making a bispecific anti-humanA-beta/human transferrin receptor antibody is provided, wherein themethod comprises culturing a host cell comprising a nucleic acidencoding the antibody, as provided above, under conditions suitable forexpression of the antibody, and optionally recovering the antibody fromthe host cell (or host cell culture medium).

For recombinant production of a bispecific anti-human A-beta/humantransferrin receptor antibody, nucleic acid encoding an antibody, e.g.,as described above, is isolated and inserted into one or more vectorsfor further cloning and/or expression in a host cell. Such nucleic acidmay be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, N.J. (2003), pp. 245-254, describing expression ofantibody fragments in E. coli.) After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHOcells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

C. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the bispecific anti-human A-beta/humantransferrin receptor antibodies provided herein are useful for detectingthe presence of A-beta in a biological sample. The term “detecting” asused herein encompasses quantitative or qualitative detection. Incertain embodiments, a biological sample comprises a cell or tissue.

In one embodiment, a bispecific anti-human A-beta/human transferrinreceptor antibody for use in a method of diagnosis or detection isprovided. In a further aspect, a method of detecting the presence ofA-beta in a biological sample is provided. In certain embodiments, themethod comprises contacting the biological sample with a bispecificanti-human A-beta/human transferrin receptor antibody as describedherein under conditions permissive for binding of the bispecificanti-human A-beta/human transferrin receptor antibody to A-beta, anddetecting whether a complex is formed between the bispecific anti-humanA-beta/human transferrin receptor antibody and A-beta. Such method maybe an in vitro or in vivo method.

In certain embodiments, labeled bispecific anti-human A-beta/humantransferrin receptor antibodies are provided. Labels include, but arenot limited to, labels or moieties that are detected directly (such asfluorescent, chromophoric, electron-dense, chemiluminescent, andradioactive labels), as well as moieties, such as enzymes or ligands,that are detected indirectly, e.g., through an enzymatic reaction ormolecular interaction. Exemplary labels include, but are not limited to,the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

D. Pharmaceutical Formulations

Pharmaceutical formulations of a bispecific anti-human A-beta/humantransferrin receptor antibody as described herein are prepared by mixingsuch antibody having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)), in theform of lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspoly(vinylpyrrolidone); amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further include interstitialdrug dispersion agents such as soluble neutral-active hyaluronidaseglycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidaseglycoproteins, such as rhuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs and methods of use, including rhuPH20, aredescribed in US 2005/0260186 and US 2006/0104968. In one aspect, asHASEGP is combined with one or more additional glycosaminoglycanasessuch as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes. In one embodiment the formulationis isotonic.

E. Therapeutic Methods and Compositions

Any of the bispecific anti-human A-beta/human transferrin receptorantibodies provided herein may be used in therapeutic methods.

In one aspect, a bispecific anti-human A-beta/human transferrin receptorantibody for use as a medicament is provided. In further aspects, abispecific anti-human A-beta/human transferrin receptor antibody for usein preventing and/or treating a disease associated with amyloidogenesisand/or amyloid-plaque formation is provided. In certain embodiments, abispecific anti-human A-beta/human transferrin receptor antibody for usein a method of treatment is provided. In certain embodiments, herein isprovided a bispecific anti-human A-beta/human transferrin receptorantibody for use in a method of treating an individual having a diseaseassociated with amyloidogenesis and/or amyloid-plaque formationcomprising administering to the individual an effective amount of thebispecific anti-human A-beta/human transferrin receptor antibody. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, such as listed below or an anti-pTau or an anti-alpha-synucleinantibody. In further embodiments, herein is provided a bispecificanti-human A-beta/human transferrin receptor antibody for use ininhibiting the formation of plaques and/or disintegrating β-amyloidplaques. In certain embodiments, herein is provided a bispecificanti-human A-beta/human transferrin receptor antibody for use in amethod of inhibiting the formation of plaques and/or disintegratingβ-amyloid plaques in an individual comprising administering to theindividual an effective of the bispecific anti-human A-beta/humantransferrin receptor antibody to inhibit the formation of plaques and/orto disintegrate β-amyloid plaques. An “individual” according to any ofthe above embodiments is preferably a human.

In a further aspect, herein is provided the use of a bispecificanti-human A-beta/human transferrin receptor antibody in the manufactureor preparation of a medicament. In one embodiment, the medicament is fortreatment of a disease associated with amyloidogenesis and/oramyloid-plaque formation. In a further embodiment, the medicament is foruse in a method of treating a disease associated with amyloidogenesisand/or amyloid-plaque formation comprising administering to anindividual having a disease associated with amyloidogenesis and/oramyloid-plaque formation an effective amount of the medicament. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, such as listed below or an anti-pTau or an anti-alpha synucleinantibody. In a further embodiment, the medicament is for the inhibitionof the formation of plaques and/or the disintegration of β-amyloidplaques. In a further embodiment, the medicament is for use in a methodof inhibiting the formation of plaques and/or the disintegration ofβ-amyloid plaques in an individual comprising administering to theindividual an amount effective of the medicament to inhibit theformation of plaques and/or to disintegrate β-amyloid plaques. An“individual” according to any of the above embodiments may be a human.

In a further aspect, herein is provided a method for treating a diseaseassociated with amyloidogenesis and/or amyloid-plaque formation. In oneembodiment, the method comprises administering to an individual having adisease associated with amyloidogenesis and/or amyloid-plaque formationan effective amount of a bispecific anti-human A-beta/human transferrinreceptor antibody. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, such as given below or an anti-pTau or ananti-alpha-synuclein antibody. An “individual” according to any of theabove embodiments may be a human.

In a further aspect, herein is provided a method for inhibiting theformation of plaques and/or for disintegrating β-amyloid plaques in anindividual. In one embodiment, the method comprises administering to theindividual an effective amount of a bispecific anti-human A-beta/humantransferrin receptor antibody to inhibit the formation of plaques and/orto disintegrate β-amyloid plaques. In one embodiment, an “individual” isa human.

In a further aspect, herein are provided pharmaceutical formulationscomprising any of the bispecific anti-human A-beta/human transferrinreceptor antibodies provided herein, e.g., for use in any of the abovetherapeutic methods. In one embodiment, a pharmaceutical formulationcomprises any of the bispecific anti-human A-beta/human transferrinreceptor antibodies provided herein and a pharmaceutically acceptablecarrier. In another embodiment, a pharmaceutical formulation comprisesany of the bispecific anti-human A-beta/human transferrin receptorantibodies provided herein and at least one additional therapeuticagent, e.g., as given below or an anti-pTau or an anti-alpha-synucleinantibody.

Antibodies as reported herein can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody as reportedherein may be co-administered with at least one additional therapeuticagent. In certain embodiments, an additional therapeutic agent is atherapeutic agent effective to treat the same or a differentneurological disorder as the bispecific antibody as reported herein isbeing employed to treat. Exemplary additional therapeutic agentsinclude, but are not limited to: the various neurological drugsdescribed above, cholinesterase inhibitors (such as donepezil,galantamine, rovastigmine, and tacrine), NMDA receptor antagonists (suchas memantine), amyloid beta peptide aggregation inhibitors,antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimicsor NGF gene therapy, PPARy agonists, HMS-CoA reductase inhibitors(statins), ampakines, calcium channel blockers, GABA receptorantagonists, glycogen synthase kinase inhibitors, intravenousimmunoglobulin, muscarinic receptor agonists, nicrotinic receptormodulators, active or passive amyloid beta peptide immunization,phosphodiesterase inhibitors, serotonin receptor antagonists andanti-amyloid beta peptide antibodies. In certain embodiments, the atleast one additional therapeutic agent is selected for its ability tomitigate one or more side effects of the neurological drug.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody as reported herein can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of thebispecific anti-human A-beta/human transferrin receptor antibody andadministration of an additional therapeutic agent occur within about onemonth, or within about one, two or three weeks, or within about one,two, three, four, five, or six days, of each other. Antibodies asreported herein can also be used in combination with otherinterventional therapies such as, but not limited to, radiation therapy,behavioral therapy, or other therapies known in the art and appropriatefor the neurological disorder to be treated or prevented.

An antibody as reported herein (and any additional therapeutic agent)can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies as reported herein would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

Lipid-based methods of transporting the fusion construct or a compoundacross the BBB include, but are not limited to, encapsulating the fusionconstruct or a compound in liposomes that are coupled to monovalentbinding entity that bind to receptors on the vascular endothelium of theBBB (see e.g., US 2002/0025313), and coating the monovalent bindingentity in low-density lipoprotein particles (see e.g., US 2004/0204354)or apolipoprotein E (see e.g., US 2004/0131692).

For the prevention or treatment of disease, the appropriate dosage of anantibody as reported herein (when used alone or in combination with oneor more other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate as reported hereinin place of or in addition to a bispecific anti-human A-beta/humantransferrin receptor antibody.

III. Articles of Manufacture

In another aspect as reported herein, an article of manufacturecontaining materials useful for the treatment, prevention and/ordiagnosis of the disorders described above is provided. The article ofmanufacture comprises a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, IV solution bags, etc. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is by itself or combined withanother composition effective for treating, preventing and/or diagnosingthe condition and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is an antibody as reported herein. The label orpackage insert indicates that the composition is used for treating thecondition of choice. Moreover, the article of manufacture may comprise(a) a first container with a composition contained therein, wherein thecomposition comprises an antibody as reported herein; and (b) a secondcontainer with a composition contained therein, wherein the compositioncomprises a further cytotoxic or otherwise therapeutic agent. Thearticle of manufacture in this embodiment may further comprise a packageinsert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate as reported herein in place of or in additionto a bispecific antibody as reported herein.

IV. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according tonumbering according to Kabat (Kabat, E. A., et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The long gene segments, which were flanked bysingular restriction endonuclease cleavage sites, were assembled byannealing and ligating oligonucleotides including PCR amplification andsubsequently cloned via the indicated restriction sites. The DNAsequences of the subcloned gene fragments were confirmed by DNAsequencing. Gene synthesis fragments were ordered according to givenspecifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described bispecific antibodies, expressionplasmids for transient expression (e.g. in HEK293 cells) based either ona cDNA organization with or without a CMV-intron A promoter or on agenomic organization with a CMV promoter can be applied.

Beside the antibody expression cassette the vectors contain:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a β-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody gene is composed of the followingelements:

-   -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   the intron A sequence in the case of cDNA organization,    -   a 5′-untranslated region derived from a human antibody gene,    -   an immunoglobulin heavy chain signal sequence,    -   the respective antibody chain encoding nucleic acid either as        cDNA or with genomic exon-intron organization,    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes encoding the antibody chains are generated by PCRand/or gene synthesis and assembled by known recombinant methods andtechniques by connection of the according nucleic acid segments e.g.using unique restriction sites in the respective vectors. The subclonednucleic acid sequences are verified by DNA sequencing. For transienttransfections larger quantities of the plasmids are prepared by plasmidpreparation from transformed E. coli cultures (Nucleobond AX,Macherey-Nagel).

For all constructs knob-into-hole heterodimerization technology was usedwith a typical knob (T366W) substitution in the first CH3 domain and thecorresponding hole substitutions (T366S, L368A and Y407V) in the secondCH3 domain (as well as two additional introduced cysteine residuesS354C/Y349′C) (contained in the respective corresponding heavy chain(HC) sequences depicted above).

Cell Culture Techniques

Standard cell culture techniques as described in Current Protocols inCell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B.,Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons,Inc., are used.

Transient Transfections in HEK293-F System

The bispecific antibodies are produced by transient expression.Therefore a transfection with the respective plasmids using the HEK293-Fsystem (Invitrogen) according to the manufacturer's instruction is done.Briefly, HEK293-F cells (Invitrogen) growing in suspension either in ashake flask or in a stirred fermenter in serum-free FreeStyle™ 293expression medium (Invitrogen) are transfected with a mix of therespective expression plasmids and 293fectin™ or fectin (Invitrogen).For 2 L shake flask (Corning) HEK293-F cells are seeded at a density of1.0*10⁶ cells/mL in 600 mL and incubated at 120 rpm, 8% CO₂. On the nextday the cells are transfected at a cell density of approx. 1.5*10⁶cells/mL with approx. 42 mL of a mixture of A) 20 mL Opti-MEM medium(Invitrogen) comprising 600 μg total plasmid DNA (1 μg/mL) and B) 20 mlOpti-MEM medium supplemented with 1.2 mL 293 fectin or fectin (2 μl/mL).According to the glucose consumption glucose solution is added duringthe course of the fermentation. The supernatant containing the secretedantibody is harvested after 5-10 days and antibodies are either directlypurified from the supernatant or the supernatant is frozen and stored.

Protein Determination

The protein concentration of purified antibodies and derivatives wasdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace, et al., Protein Science 4 (1995) 2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants was estimated by immunoprecipitation with protein Aagarose-beads (Roche Diagnostics GmbH, Mannheim, Germany). Therefore, 60μL protein A Agarose beads were washed three times in TBS-NP40 (50 mMTris buffer, pH 7.5, supplemented with 150 mM NaCl and 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant was applied to theprotein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 hour at room temperature the beads were washed on anUltrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche Diagnostics GmbH,Mannheim, Germany) and briefly four times with 0.5 mL 100 mM Na-citratebuffer (pH 5.0). Bound antibody was eluted by addition of 35 μl NuPAGE®LDS sample buffer (Invitrogen). Half of the sample was combined withNuPAGE® sample reducing agent or left unreduced, respectively, andheated for 10 min at 70° C. Consequently, 5-30 μl were applied to a4-12% NuPAGE® Bis-Tris SDS-PAGE gel (Invitrogen) (with MOPS buffer fornon-reduced SDS-PAGE and MES buffer with NuPAGE® antioxidant runningbuffer additive (Invitrogen) for reduced SDS-PAGE) and stained withCoomassie Blue.

The concentration of the antibodies in cell culture supernatants wasquantitatively measured by affinity HPLC chromatography. Briefly, cellculture supernatants containing antibodies that bind to protein A wereapplied to an Applied Biosystems Poros A/20 column in 200 mM KH₂PO₄, 100mM sodium citrate, pH 7.4 and eluted with 200 mM NaCl, 100 mM citricacid, pH 2.5 on an Agilent HPLC 1100 system. The eluted antibody wasquantified by UV absorbance and integration of peak areas. A purifiedstandard IgG1 antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants was measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Streptavidin A-96 well microtiter plates (RocheDiagnostics GmbH, Mannheim, Germany) were coated with 100 μL/wellbiotinylated anti-human IgG capture molecule F(ab′)2<h-Fcγ>BI (Dianova)at 0.1 μg/mL for 1 hour at room temperature or alternatively overnightat 4° C. and subsequently washed three times with 200 μL/well PBS, 0.05%Tween (PBST, Sigma). Thereafter, 100 μL/well of a dilution series in PBS(Sigma) of the respective antibody containing cell culture supernatantswas added to the wells and incubated for 1-2 hour on a shaker at roomtemperature. The wells were washed three times with 200 μL/well PBST andbound antibody was detected with 100 μl F(ab′)2<hFcγ>POD (Dianova) at0.1 μg/mL as the detection antibody by incubation for 1-2 hours on ashaker at room temperature. Unbound detection antibody was removed bywashing three times with 200 μL/well PBST. The bound detection antibodywas detected by addition of 100 μL ABTS/well followed by incubation.Determination of absorbance was performed on a Tecan Fluor Spectrometerat a measurement wavelength of 405 nm (reference wavelength 492 nm).

Preparative Antibody Purification

Antibodies were purified from filtered cell culture supernatantsreferring to standard protocols. In brief, antibodies were applied to aprotein A Sepharose column (GE healthcare) and washed with PBS. Elutionof antibodies was achieved at pH 2.8 followed by immediateneutralization. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine buffer comprising 150 mM NaCl(pH 6.0). Monomeric antibody fractions were pooled, concentrated (ifrequired) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugalconcentrator, frozen and stored at −20° C. or −80° C. Part of thesamples were provided for subsequent protein analytics and analyticalcharacterization e.g. by SDS-PAGE, size exclusion chromatography (SEC)or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

CE-SDS

Purity and antibody integrity were analyzed by CE-SDS using microfluidicLabchip technology (PerkinElmer, USA). Therefore, 5 μl of antibodysolution was prepared for CE-SDS analysis using the HT Protein ExpressReagent Kit according manufacturer's instructions and analyzed onLabChip GXII system using a HT Protein Express Chip. Data were analyzedusing LabChip GX Software.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄ buffer(pH 7.5) on an Dionex Ultimate® system (Thermo Fischer Scientific), orto a Superdex 200 column (GE Healthcare) in 2×PBS on a DionexHPLC-System. The eluted antibody was quantified by UV absorbance andintegration of peak areas. BioRad Gel Filtration Standard 151-1901served as a standard.

Mass Spectrometry

This section describes the characterization of the bispecific antibodieswith emphasis on their correct assembly. The expected primary structureswere analyzed by electrospray ionization mass spectrometry (ESI-MS) ofthe deglycosylated intact antibody and in special cases of thedeglycosylated/limited LysC digested antibody.

The antibodies were deglycosylated with N-Glycosidase F in a phosphateor Tris buffer at 37° C. for up to 17 h at a protein concentration of 1mg/ml. The limited LysC (Roche Diagnostics GmbH, Mannheim, Germany)digestions were performed with 100 μg deglycosylated antibody in a Trisbuffer (pH 8) at room temperature for 120 hours, or at 37° C. for 40min, respectively. Prior to mass spectrometry the samples were desaltedvia HPLC on a Sephadex G25 column (GE Healthcare). The total mass wasdetermined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik)equipped with a TriVersa NanoMate source (Advion).

Chemical Degradation Test

Samples were split into three aliquots and re-buffered into 20 mMHis/His*HCl, 140 mM NaCl, pH 6.0 or into PBS, respectively, and storedat 40° C. (His/NaCl) or 37° C. (PBS). A control sample was stored at−80° C.

After incubation ended, samples were analyzed for relative activeconcentration (BIAcore), aggregation (SEC) and fragmentation (capillaryelectrophoresis or SDS-PAGE) and compared with the untreated control.

Thermal Stability

Samples were prepared at a concentration of 1 mg/mL in 20 mMHistidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into anoptical 384-well plate by centrifugation through a 0.4 μm filter plateand covered with paraffin oil. The hydrodynamic radius was measuredrepeatedly by dynamic light scattering on a DynaPro Plate Reader (Wyatt)while the samples were heated with a rate of 0.05° C./min from 25° C. to80° C.

Alternatively, samples were transferred into a 10 μL micro-cuvette arrayand static light scattering data as well as fluorescence data uponexcitation with a 266 nm laser were recorded with an Optim1000instrument (Avacta Inc.), while they were heated at a rate of 0.1°C./min from 25° C. to 90° C.

The aggregation onset temperature is defined as the temperature at whichthe hydrodynamic radius (DLS) or the scattered light intensity(Optim1000) starts to increase.

Alternatively, samples were transferred in a 9 μL multi-cuvette array.The multi-cuvette array was heated from 35° C. to 90° C. at a constantrate of 0.1° C./minute in an Optim1000 instrument (Avacta AnalyticalInc.). The instrument continuously records the intensity of scatteredlight of a 266 nm laser with a data point approximately every 0.5° C.Light scattering intensities were plotted against the temperature. Theaggregation onset temperature (T_agg) is defined as the temperature atwhich the scattered light intensity begins to increase.

The melting temperature is defined as the inflection point influorescence intensity vs. wavelength graph

Example 1

Expression and Purification

The bispecific antibodies were produced as described above in thegeneral materials and methods section.

The bispecific antibodies were purified from the supernatant by acombination of protein A affinity chromatography and size exclusionchromatography. The obtained products were characterized for identity bymass spectrometry and analytical properties such as purity by CE-SDS,monomer content and stability.

The expected primary structures were analyzed by electrospray ionizationmass spectrometry (ESI-MS) of the deglycosylated intact antibody anddeglycosylated/plasmin digested or alternatively deglycosylated/limitedLysC digested antibody as described in the general methods section.

Additional analytical methods (e.g. thermal stability, mass spectrometryand functional assessment) were only applied after protein A and SECpurification.

Example 2

Determination of Binding to Aβ1-40 Fibers In Vitro by ELISA

Binding of the bispecific antibodies to fibrillar Aβ is measured by anELISA assay. Briefly, Aβ(1-40) is coated at 7 μg/mL in PBS onto Maxisorbplates for 3 days at 37° C. to produce fibrillar Abeta, and then driedfor 3 h at RT. The plate is blocked with 1% CroteinC and 0.1% RSA in PBS(blocking buffer) for 1 h at RT, then washed once with wash buffer.Bispecific antibodies or controls are added at concentrations up to 100nM in blocking buffer and incubated at 4° C. overnight. After 4 washsteps, constructs are detected by addition of anti-human-IgG-HRP(Jackson Immunoresearch) at 1:10,000 dilution in blocking buffer (1 RT),followed by 6 washes and incubation in TMB (Sigma). Absorbance is readout at 450 nm after stopping color development with 1 N HCl.

Example 3

Determination of Binding to Transferrin Receptor In Vitro

Binding of the bispecific antibodies to murine transferrin receptor istested by FACS analysis on mouse X63.AG8-563 myeloma cells. If the Aβantibody shows a certain tendency to non-specifically bind to Ag8 cells,specific binding can be quantified by co-incubation with a 20 foldexcess of anti-mouse-TfR antibody. Cells are harvested bycentrifugation, washed once with PBS and 5×10⁴ cells incubated with a1.5 pM to 10 nM dilution series of the polypeptide fusions with orwithout addition of 200 nM anti-mouse TfR antibody in 100 μL RPMI/10%FCS for 1.5 h on ice. After 2 washes with RPMI/10% FCS, cells areincubated with goat-anti-human IgG coupled to Phycoerythrin (JacksonImmunoresearch) at a dilution of 1:600 in RPMI/19% FCS for 1.5 h on ice.Cells are again washed, resuspended in RPMI/10% FCS and Phycoerythrinfluorescence measured on a FACS-Array instrument (Becton-Dickinson).

Example 4

Surface Plasmon Resonance-Based Binding Assay for Human TfR-AntibodyInteraction

The binding experiment were carried out on a BIAcore B 4000 (GEHealthcare) equipped with C1 sensor chip (GE Healthcare, cat.no.BR1005-35) pre-treated with anti-human Fab antibody (GE Healthcare,cat.no 28-9583-25) using a standard amine coupling chemistry procedureaccordingly to the vendor's manual.

For kinetic measurements the sample antibody was immobilized applying acontact time of 60 seconds and a flow rate of 10 μL/min in phosphatebuffer saline pH 7.4, 0.05% Tween 20 at 25° C. Recombinant His6-taggedhuman transferrin receptor (R&D systems, cat.no 2474-TR-050) was appliedin increasing concentrations and the signal monitored over the time. Anaverage time span of 150 seconds of association time and 600 seconds ofdissociation time at 30 μL/min flow rate was recorded. Data were fitusing a 1:1 binding model (Langmuir isotherm).

Example 5

Staining of Native Human β-amyloid Plaques from Brain Sections of anAlzheimer's Disease Patient by Indirect Immunofluorescence using aBispecific Antibody as Reported Herein

The bispecific antibodies can be tested for the ability to stain nativehuman β-amyloid plaques by immunohistochemistry analysis using indirectimmunofluorescence. Specific and sensitive staining of genuine humanβ-amyloid plaques can be demonstrated. Cryostat sections of unfixedtissue from the temporal cortex obtained postmortem from patientspositively diagnosed for Alzheimer's disease are labeled by indirectimmunofluorescence. A two-step incubation is used to detect boundbispecific antibody, which is revealed by affinity-purified goatanti-human (GAH555) IgG (H+L) conjugated to Alexa 555 dye (MolecularProbes). Controls can include unrelated human IgG1 antibodies (Sigma)and the secondary antibody alone, which all should give negativeresults.

Example 6

In Vivo β-amyloid Plaque Decoration by a Bispecific Antibody as ReportedHerein in a Mouse Model of Alzheimer's Disease

Bispecific antibody can be tested in APP/PS2 double transgenic mice, amouse model for AD-related amyloidosis (Richards, J. Neuroscience, 23(2003) 8989-9003) for their ability to immuno-decorate β-amyloid plaquesin vivo. This enabled assessment of the extent of brain penetration andbinding to amyloid-β plaques. The fusion polypeptide can be administeredat different doses compared to naked anti-Aβ monoclonal antibody andafter 6 days animals are perfused with phosphate-buffered saline and thebrains frozen on dry ice and prepared for cryosectioning.

The presence of the antibodies bound to β-amyloid plaques can beassessed using unfixed cryostat sections either by single-labeledindirect immunofluorescence with goat anti-human IgG (H+L) conjugated toAlexa555 dye (GAH555) (Molecular Probes) at a concentration of 15 μg /mlfor 1 hour at room temperature. A counterstaining for amyloid plaquescan be done by incubation with BAP-2, a mouse monoclonal antibodyagainst Aβ conjugated to Alexa 488 at a concentration of 0.5 μg/ml for 1hour at room temperature. Slides are embedded with fluorescence mountingmedium (S3023 Dako) and imaging is done by confocal laser microscopy.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

The invention claimed is:
 1. A bispecific antibody specifically bindingto human A-beta protein and human transferrin receptor, comprising: afirst light chain of SEQ ID NO: 01 or that has a sequence identity toSEQ ID NO: 01 of at least 90%; a first heavy chain of SEQ ID NO: 02 orthat has a sequence identity to SEQ ID NO: 02 of at least 90%; a secondlight chain of SEQ ID NO: 03 or that has a sequence identity to SEQ IDNO: 03 of at least 90%; and a second heavy chain of SEQ ID NO: 10 orthat has a sequence identity to SEQ ID NO: 10 of at least 90%, whereinsubstitutions, insertions, or deletions occur in regions outsidehypervariable regions.
 2. The bispecific antibody according to claim 1,wherein the bispecific antibody is monoclonal.
 3. A pharmaceuticalformulation, comprising the bispecific antibody according to claim 1 anda pharmaceutically acceptable carrier.
 4. A method of treatingAlzheimer's disease in an individual, comprising the step ofadministering an effective amount of the bispecific antibody accordingto claim 1 to said individual in need thereof.
 5. A method of inhibitingor slowing down the formation of plaques in the brain of an individual,comprising the step of administering an effective amount of thebispecific antibody according to claim 1 to said individual in needthereof.